The invention concerns microbiology and medicine and may be used for the prophylaxis and treatment of the syndrome of an irritated large intestine in either a human or an animal.
Having the entire spectrum of normal intestine microflora is a necessary link for many metabolic reactions in humans, particularly for eliminating pathogenic microorganisms in the gastrointestinal tract. The microflora participates in the hepatoenteric circulation of the main components of bile; the flora ferments in the distal section of the intestine and inactivates biologically active compounds (biogenic amines), which are discharged together with the digestive juices; the intestinal flora utilizes the non-digested alimentary substances with the formation of amines, organic acids and other compounds which influence the metabolism of the organism.
One of the important functions of the normal intestinal microflora is its participation in the immunological reactivity of macroorganisms. The total reserve of immunoglobins is created as the result of antigenic stimulation by autoflora, most importantly, by the intestinal bacteria.
Methods are known for the prophylaxis of the syndrome of an irritated large intestine in newborns based on the introduction of strains of bifidobacteria from the mother microbial corpus in a dose of 1xc3x97108 to 1xc3x97109 microbial cells into the anal orifice 30-120 minutes after birth (RF Certificate No. 1743607, 1992). This method is effective only when delivery is done by cesarean section. But for the prophylaxis of the syndrome of an irritated large intestine, the entire spectrum of the healthy intestinal microflora is not used, but only a monoculture of bifidobacteria. Besides, the given culture of the bacteria is not adapted to the intestine of the newborn and may not be acclimatized to the intestine, because it is not taken from the newborn intestine (not autostrain). Thus, this method of prophylaxis is not effective enough. When introduced anally, a part of the bacteria may die while passing through the various sections of the gastrointestinal tract.
Methods for creation and use of a complex bacteria preparation for correction of intestinal disbiosis are known, consisting of sampling from the human and making a biomass of autostrains of lactobacteria or Escherichia coli with the following combination of microorganisms as a base. Active carbon can serve as a support. The microbial cells are supported on the surface of the active carbon particles in a concentration of 2.1xc3x97103 to 1.0xc3x97105 cells/mm2 of carbon surface area. The active carbon is utilized as a powder with a maximum particle size of 30 microns. The preparation is inserted into the anal orifice of the human three times per every 3-4 days in a dose of 30-100 microbial cells per gram (RF Certificate No. 2017486, 1994). But for the prophylaxis of the syndrome of irritated large intestine, the entire spectrum of the human intestine is not used, but only a monoculture of lactobacteria or Escherichia coli. This fact leads to a decreased efficiency for the prophylaxis procedure. A treatment method for the syndrome of irritated large intestine in humans is known, consisting of the following stages: sampling of feces, dilution of the material to be studied using a sterile physiological solution, its inoculation in various dilutions onto a culture medium, identification, selection of autostrains of normal intestinal microflora (bidifo- and lactobacteria), separate preparation of cultures of each kind of bacteria, mixing the cultures, short-term storage at a temperature of +4 to 7xc2x0 C., and periodic introduction of the mixture of the given autostrains into the human intestine during the treatment of the disease, but after antibiotic therapy (RF Certificate No. 1286212, 1987).
The main drawback to the known treatment methods for the syndrome of irritated large intestine lies in the use of only the autostrains of bifido- and lactobacteria rather than the entire spectrum of healthy human microflora. Thus, the efficiency of the treatment is decreased. Besides, the selection of autostrains is carried out before antibiotic therapy, in the period when the normal (healthy) microflora of a man is in the xe2x80x9coppressedxe2x80x9d state. When introduced anally, a part of the bacteria may die while passing through the gastrointestinal tract: the efficiency of the treatment of the syndrome of irritated large intestine is decreased, and the recovery time is prolonged. We offer a method for creation of a bank of autochthonous strains of microorganisms for the recovery of the intestinal microbiocenosis in humans with a stable treatment-and-prophylactic effect, achieved by simultaneously influencing various links of the broken bacteriocenosis of the human intestine by use of the entire spectrum of the normal (healthy) microflora of the intestine.
According to the main purpose of the given invention, the following method is offered: feces samples of a single human are taken during healthy periods beginning 7-15 days after birth and continuing through the course of his entire lifetimexe2x80x94periodically, not more often than once a year. The autostrains of the normal intestinal microflora are selected from the feces and identified, a culture of each kind of bacteria is separately made on a selective culture medium to a minimum titer of 103-109 cells/ml: the biomasses are mixed and a special stabilizer is added; the mixture is divided into separate samples, which are conserved and stored during the entire lifetime of the man with periodic testing of the biotiter.
At the 7th-15th day after birth of a human the formation of the intestinal microflora is complete.
The use of a bank of autochthonous strains of microorganisms including the entire spectrum of the normal intestinal microflora of a human ensures the complex biological influence (when the mixture is introduced into the intestine) directed to the generation of normal microbiocenosis.
In the baby stage (up to 71 year) we select as the normal intestinal microflora: 1. the following kinds of bifidobacteria: Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis; and 2. the following kinds of lactobacteria: Lactobacillus acidophilus, Lactobacillus fermenti.
From the age of 1 year and older we select as the normal intestinal microflora as a rule the bifidobacteria of the types Bifidobacterium longum, Bifidobacterium adolescentis, the lactobacteria of the types Lactobacillus acidophilus, Lactobacillus fermenti, Lactobacillus plantarum, the strains of bacteria Escherichia coli and Streptococcus faecium, Streptococcus faecalis, Streptococcus avium, Streptococcus salivarius and Streptococcus bovis.
In order to create a bank of autochthonous strains, it""s necessary to provide samples of strains from various periods of lifexe2x80x94it ensures a wide spectrum and complex influence.
Each kind of culture is blended together in equal proportion: the loss of biological activity of the various strains in the mixture during the conservation and storage is decreased.
The autostrain culture mixture is stabilized with saccharose-gel or saccharose-starch protection medium at 5-10 mass %. When the concentration of stabilizer decreases, biological activity in the culture decreases during drying and subsequent storage.
The culture samples are preserved through lyophilization.
During the storage period a part of the samples of bacteria autostrains from various periods of life are mixed in equal proportions after biotiter controlxe2x80x94this increases the biological activity of the preparation and the acclimatization of the bacteria in the human intestine.
The use of the entire spectrum of the normal (healthy) autochthonous microflora of the intestine permits repair of the broken bacteriocenosis of the intestine in the process of the treatment of the syndrome of irritated large intestine or to prevent the possible breach of bacteriocenosis during the treatment of this disease.
In order to form a bank of autochthonous strains of microorganisms, samples of human feces are obtained not earlier than the 7th-15th day after the birth. The selected material is repeatedly diluted by a factor of 10 with sterile physiological solution to a dilution of 108. The material from various solutions is inoculated onto a culture medium of Blaurock, Sabur, Endo, Kalina, MRS-4, or blood agar with polymyxin for determination of the initial levels of Escherichia coli and the identification and selection of autostrains of the normal intestinal microflora, from the baby stage, consisting of bidifo- and lactobacteria.
The material from the isolated colonies of lactobacteria (Lactobacillus acidophilus, Lactobacillus fermenti) is cultured on the medium MRS-4, and the isolated colonies of bifidobacteria (Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis) are cultured on the medium of Blaurock. After two days, the cultures are inoculated a second time onto the elective culture medium. After culturing the second time for two days, the colonies of bifidobacteria and lactobacteria are inoculated (separately) onto the above-mentioned mediums of Blaurock and MRS-4 and cultured according to standard methods of production of biomass. For example, the biomass of bifidobacteria (Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis) has a titer of not less than 109 cells/ml, the biomass of lactobacteria (Lactobacillus acidophilus, Lactobacillus fermenti)xe2x80x94not less than 1091010 cells/ml.
Equal quantities of each kind of the autochthonous strains of the microorganisms are placed into the reactor and mixed. About 5-10 mass% saccharose-gel protection medium (stabilizer) is added to the biomass.
The biomass is then poured into ampullae and dried by standard lyophilization, sealed, and stored at a temperature of not more than xe2x88x9218xc2x0 C. in order to ensure the activity of the preparation for a long time period, for example, a human lifetime.
Periodically (not less than once a year), the biotiter of one or several samples of the bank is tested by inoculation onto a culture medium specific for the autostrains"" mixture and by bacterial counts of each strain. If the biotiter of the stored preparation declines, the entire volume of biomass is activated by culturing on the specific medium until the biotiter reaches the initial level. Thereafter stabilizer is added to the biomass, and storage is continued at a maximum temperature of xe2x88x9218xc2x0 C.
The second selection of feces samples from the same person is carried out at the age of 1-2 years, and then at the age of 4-5 years and so on throughout the entire lifespan. The processing of the biomass mixture is similar to the above-mentioned method. The difference consists in the changing composition of the normal intestinal microflora as a function of age in the children and adults, as well as in the stabilization of the number of bifidobacteria at the level of 108-1010 microbial cells/g of feces. The Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis, which predominate in young children, are replaced with Bifidobacterium longum, Bifidobacterium adolescentis etc., which are common in juveniles and adults.
The material to be studied is repeatedly diluted ten-fold with sterile physiological solution to a level of 10xe2x88x928. The material from various stages of dilution is inoculated onto the culture media of Blaurock, Sabur, Endo, Kalina, MRS-4, or blood agar with polymyxin for determination of the initial levels of Escherichia coli, lactobacteria, Enterococcus, Staphylococcus and other bacteria of the following types followed by selection of autostrains of the normal intestinal microflora consisting of bifidobacteria, lactobacteria, Escherichia coli, milk streptococcus, and Lactobacillus. The isolated colonies of lactobacteria (Lactobacillus acidophilus, Lactobacillus fermenti, Lactobacillus plantarum) are cultured on the medium MRS-4, the isolated colonies of bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis) on the medium Blaurock, the isolated colonies of enterococcus (Streptococcus faecium, Streptococcus faecalis, Streptococcus avium, Streptococcus salivarius and Streptococcus bovis) on the medium Kalina, and the isolated colonies of Escherichia coli on blood agar with polymyxin. After culturing for two days, they are inoculated once more onto the corresponding selective culture medium. The colonies are cultured separately on the culture media of Blaurock, MRS-4, Kalina, or blood agar with polymyxin by standard methods to increase the biomass. For example, the biomass of bifidobacteria has a minimum titer of 109 cells/ml, lactobacteria a minimum of 1091010 cells/ml, Escherichia coli a minimum of 107 108 cells/ml, and Streptococcus faecium a minimum of 109 cells/ml.
Then each type of microorganism is sampled in equal proportions and mixed in the reactor. Saccharose-starch protective medium is added to the biomass of the autostrains at a level of 5-10 mass% of the biomass. The conservation, storage and testing (control) of the autostrains are similar to the technology described above (in the case of newborn). The samples of the mixture are stored for a human lifetime.
Periodically, feces samples are taken from the same man between the ages of 30-45 years and older during the period when he is healthy and in normal intestinal function. The technology of obtaining, preserving, and storing the samples is described above. The samples are stored for the lifetime of this human.
After adjusting the biotiter during storage, the samples of normal intestinal microflora in various periods of the lifetime of the human and prepared in compliance with the above-mentioned technology are mixed in equal proportions. This insures the biological activity of the mixture of newly received samples of autostrains and the acclimatization of the autostrains in the human intestine.
The totality of stored preparations obtained according to the described technology provides the BANK OF AUTOCHTHONOUS STRAINS OF MICROORGANISMS which may be used for the recovery of the intestinal microbiocenosis of the man.
USE OF THE BANK OF AUTOCHTHONOUS STRAINS OF MICROORGANISMS IN THE PRACTICE OF THE TREATMENT OF VARIOUS DISEASES CONNECTED WITH AN IRRITATED LARGE INTESTINE
Diseases based on the syndrome of an irritated large intestine are treated by recovery of the normal intestinal biocenosis with the help of preparations manufactured from the microorganisms of the normal human microflora. These diseases include, for example, allergic dermatoses, the syndrome of chronic fatigue, bronchial asthma etc. There are diseases, for which a complex treatment is needed, taking into consideration the character of the course of the disease, pathogenesis, and possible complications aggravating the base disease. For example, the therapeutic treatment of the above-mentioned diseases may include pharmaceutical agents such as antibiotics, glucocorticoids, or hormones which may cause severe intestinal disorders. In such cases we find it necessary to ensure the physiological sensitization of the human by correcting the microbiocenosis of the large intestine as the therapeutic base of the main illness, thereby increasing the length of the remission period.
The syndrome of irritated large intestine may appear as the base disease, or some severe diseases may be accompanied by it.
1. Syndrome of Irritated Large Intestine
To treat this disease, one ampulla containing a sample of a mixture of autostrains prepared in accordance with the above described technology was opened, and the mixture of autostrains was activated on a differential culture medium in a thermostat for 26 hours at a temperature of +37xc2x0xc2x11xc2x0 C. Thereby the bacteria were acclimatized to the culture conditions. The received culture was used to inoculate a second preparation of the same culture medium in an amount of 5% of the total mass of the culture medium. The received culture medium was incubated at +37xc2x0xc2x11xc2x0 C. until the logarithmic phase of bacteria growth (the 2nd phase), corresponding to the maximal speed of bacteria division, which takes place in the range of 26-38 hours. At this point, there were 107 108 cells in 1 ml of received solution.